Abstract

Development of the αβ and γδ T cell lineages is dependent upon the rearrangement and expression of the TCRα and β or γ and δ genes, respectively. Although the timing and sequence of rearrangements of the TCRα and TCRβ loci in adult murine thymic precursors has been characterized, no similar information is available for the TCRγ and TCRδ loci. In this report, we show that approximately half of the total TCRδ alleles initiate rearrangements at the CD44highCD25+ stage, whereas the TCRβ locus is mainly in germline configuration. In the subsequent CD44lowCD25+ stage, most TCRδ alleles are fully recombined, whereas TCRβ rearrangements are only complete on 10–30% of alleles. These results indicate that rearrangement at the TCRδ locus can precede that of TCRβ locus recombination by one developmental stage. In addition, we find a bias toward productive rearrangements of both TCRδ and TCRγ genes among CD44highCD25+ thymocytes, suggesting that functional γδ TCR complexes can be formed before the rearrangement of TCRβ. These data support a model of lineage commitment in which sequential TCR gene rearrangements may influence αβ/γδ lineage decisions. Further, because TCR gene rearrangements are generally limited to T lineage cells, these analyses provide molecular evidence that irreversible commitment to the T lineage can occur as early as the CD44highCD25+ stage of development.

The majority of adult peripheral T cells derive from a small number of precursor cells that immigrate to the thymus from the bone marrow. During intrathymic development, these precursors expand by a million fold, differentiate into two T cell and at least two non-T cell lineages, and, within the T cell lineage, acquire the capacity to express the Ag-specific TCR complex 1 . The two T cell lineages are designated αβ and γδ, depending upon expression of the respective TCRs 2, 3 . Expression of the α-, β-, γ-, and δ-chains of the TCR requires somatic recombination of the V, D, and J genes encoding the V domain of the corresponding TCR proteins 2, 3, 4 . Because the TCR is thought to play an important role in the maturation, expansion, and possibly lineage decisions of T cells 5, 6, 7 , the relationship of intrathymic differentiation and proliferation to TCR gene recombination has been the subject of considerable interest (reviewed in 8 .

The successive steps of immature, CD4−/CD8− thymocyte differentiation have been characterized by surface expression of CD44 in the absence (stage 1) or presence (stage 2) of CD25 followed by an initial down-regulation of CD44 (stage 3) and subsequently of CD25 (stage 4) 9 . Stages 1 and 2 have been shown to have the TCRβ and TCRγ loci in germline configuration 10, 11 . Both partial (D to J) and complete (V to DJ) rearrangements of the TCRβ locus as well as V to J rearrangements of the TCRγ locus occur primarily in stage 3 10, 12 . No further rearrangements of these loci (β, γ, and δ) are known to occur after the transition to stage 4, which marks the onset of TCRα locus recombination and commitment to the αβ lineage 13 .

Stage 1 precursors can give rise to multiple lineages, including both T lineages 8 , NK cells 14 , and dendritic cells (DCs)3, 15 , but not B cells or myeloid cells 8 . Functional assays indicate that stage 3 thymocytes can no longer give rise to non-T lineage cells 1 , although they retain the bipotential capacity to develop into both αβ and γδ T cells 16 . Consequently, these studies illustrate the progressive loss of multilineage potential during thymocyte differentiation. However, the point of irreversible commitment within the T lineages (i.e., αβ or γδ) has not been determined. Furthermore, the relationship between individual TCR gene rearrangements and αβ/γδ lineage divergence has not been established. To address these questions, we characterized the association of specific TCR gene rearrangements to cellular differentiation in the adult murine thymus. Because non-T lineages (i.e., B cells, NK cells, and DCs) do not undergo TCR gene rearrangements 1, 17 , we reasoned that initiation of TCR locus recombination would mark irreversible commitment to the T cell lineage. Furthermore, such studies would be expected to provide additional insights into the relationship between TCR expression and αβ/γδ lineage decision. Our results indicate that TCRδ gene rearrangements on most alleles initiate during stage 2, suggesting that T lineage commitment can occur as early as this stage. Analysis of the relative distribution of productive TCRγ and TCRδ rearrangements further suggests that commitment to the γδ lineage can occur one developmental stage earlier than that of αβ lineage cells.

Materials and Methods

Animals

For the preparation of triple-negative (TN) thymocytes and peripheral lymph node (LN) B and T cells, C57BL/6 mice were used; these mice were originally purchased from the Jackson Laboratory (Bar Harbor, ME) and maintained at the Animal Facilities of Memorial Sloan Kettering Cancer Center. TCRβ-deficient and SCID mutant mice were purchased from the Jackson Laboratory.

Cell preparation and flow cytometric analyses

Preparation of thymocytes lacking CD3/CD4/CD8 expression was performed as described previously 18 . Briefly, CD3+/CD4+/CD8+ cells from pools of 20 freshly isolated thymi were depleted by two sequential rounds of treatment with mAbs specific for each of these proteins followed by anti-Ig-coated paramagnetic beads. CD3−/CD4−/CD8− cells were then stained with fluorochrome-conjugated mAbs specific for CD24, CD25, and CD44 and purified by cell sorting. For analysis of TCRγδ expression on early thymocyte precursors, CD3 was omitted from the depletion steps; Ab specific for TCRγδ (GL3) was included in the immunofluorescent staining. LN B and T cells from individual mice were purified by cell sorting after staining with fluorochrome-conjugated mAbs specific for CD24 and TCRβ, respectively.

Nucleic acid preparation and hybridization

High-m.w. DNA was prepared from total single-cell suspensions embedded in low melting point agarose plugs 12 , EcoRI digested, and Southern blotted 18 . The same membrane hybridized to TCRβ locus-specific probes and published previously 18 was hybridized first to a mixture of radiolabeled probe 4 and recombination-activating gene (RAG)-1 19 , followed by hybridization to a mixture of Vγ4- and Vγ7-specific probes. The Vγ probes were generated by PCR amplification of C57BL/6 kidney DNA with the following primers: 5Vγ4, GGGGATCCAACCTGGCAGATGAGA; 3Vγ4, TCTGGATCCAAGGAATATATTGTCA; 5Vγ7, CTCGGATCCTACTTCTAGCTTTCT; and 3Vγ7, GCGGATCCAGGAGGCACAGTAGTA. Rehybridization using only the Vγ4 probe (data not shown) unambiguously identified the two polymorphic germline and rearranged bands that are marked as “A” and “B” on Fig. 2⇓. Quantitative analysis was performed on a PhosphorImager using ImageQuant 3.0 software (Molecular Dynamics, Sunnyvale, CA).

PCR analysis

High-m.w. DNA prepared from various thymocyte populations 20 was amplified for 30 cycles with Taq DNA polymerase in a thermocycler using V gene-specific forward and J gene-specific reverse primers 19, 21 . The following primers were used to amplify TCRβ locus rearrangements: 5Vβ8, GCATTCTAGATGGTCCCAAGATGGGC; 5Dβ1, GTGAATTCTTCCAGCCCTCAA; and 3Jβ1.6, GGCGAATTCCAAAGGACAATGGTCCC. The PCR products were separated by sieving agarose electrophoresis, Southern blotted, and hybridized to radiolabeled oligonucleotide probes identical with the specific 3′ primer sequences. Parallel reactions with nonlymphoid kidney DNA also ensured that the bands detected in thymocyte subsets were specific for TCR gene rearrangements (data not shown). The nonrearranging RAG-2 gene was amplified with primers 22 that generate an ∼1600-bp product; this product is expected to amplify less efficiently than the smaller rearranged TCR gene products.

Results

Rearrangement of the TCRβ, TCRγ, and TCRδ loci in adult TN thymocytes

Previously, we have described quantitative Southern blot assays that can accurately measure the extent of partial D to Jδ or D to Jβ rearrangements and full V to DJδ or V to DJβ rearrangements in murine thymocytes 18, 19 . Highly purified CD3, CD4, and CD8 TN thymocytes from adult mice were electronically sorted into four subsets according to the surface expression of the CD24, CD25, and CD44 proteins 9 as described previously 18 . Genomic DNA was digested with EcoRI, Southern blotted, and hybridized sequentially to the following probes: a Vβ-specific probe proximal to the D/Jβ cluster (see Ref. 18 for results); a mixture of probe 4, which is specific to sequences between Jδ1 and Jδ2 19 and RAG-1, a probe that detects a nonrearranging gene and serves as a control for DNA loading differences (Fig. 1⇓); and finally a mixture of probes corresponding to the Vγ4 and Vγ7 genes (Fig. 2⇓).

Rearrangement of the TCRδ locus in TN thymocytes. Southern blot analysis of EcoRI-digested genomic DNA hybridized with probe 4 and RAG-1 (19). DNA prepared from LN B cells (lane 1) and from heat stable Ag+, immature TCRβ-deficient thymocytes (Thy β−/−, lane 7) served as a control for the intact germline hybridization signal and for the TCRδ locus recombination pattern in immature thymocytes, respectively. Stages 1–4 of wild-type, TN thymus subsets (lanes 2–5) and mature LN T cells (lane 6) are shown. The arrows on the sides indicate the position of known D to Jδ1 or V to DJδ1 rearrangements as determined previously (19) as well as the position of the control, nonrearranging hybridization probe, RAG-1. Stage 2 TN thymocytes have significant partial (D2 to Jδ1) and few complete (V to DJδ1) rearrangements (lane 3). Stage 3 and 4 thymocytes display TCRδ locus recombination that is as extensive as that seen for mature T cells or TCRβ deficient-thymocytes (compare lanes 4 and 5 with lanes 6 and 7).

Rearrangement of the TCRγ1 locus in TN thymocytes. The Southern blot shown on Fig. 1⇑ was rehybridized with probes specific for Vγ4 and Vγ7. DNA prepared from LN B cells (lane 1) and a SCID kidney (K Scid, lane 8) was used as a control for the intact germline hybridization signal. DNA from LN T cells (lane 6) and from heat stable Ag+, immature TCRβ-deficient thymocytes (Thy β−/−, lane 7) served as a control for the TCRγ locus recombination pattern in T cells. The arrows on the sides indicate the position of the germline fragments and Vγ to Jγ1 rearrangements. Note the presence of an RFLP (marked as A and B) that has been assigned to the Vγ4 gene by separate hybridization to the Vγ4 probe only (data not shown). Stage 2 TN thymocytes have a few complete Vγ4 to Jγ1 rearrangements, but no Vγ7 to Jγ1 rearrangements (lane 3). Stage 3 and 4 TN thymocytes have TCRγ locus recombination progressively approaching the pattern seen in mature T cells or TCRβ-deficient thymocytes (compare lanes 4 and 5 with lanes 6 and 7).

The results of TCRβ locus analysis have been published previously 18 . The results of probe 4 and RAG-1 hybridization are shown on Fig. 1⇑. In non-T cell samples (such as LN B cells), probe 4 detects a single germline EcoRI fragment (Fig. 1⇑, lane 1). In contrast, in mature LN T cells or in TCRβ-deficient immature thymocytes, virtually no germline hybridization signal can be observed, whereas numerous non-germline (i.e., rearranged) bands appear (Fig. 1⇑, lanes 6 and 7); these bands can be assigned to specific partial (D-Jδ) or complete (V-DJδ) rearrangements (Fig. 1⇑ and 19 . The least mature population of TN thymocytes (stage 1) contains >95% of the TCRδ alleles in germline configuration as determined by quantitative comparison with a non-T cell sample (Fig. 1⇑, compare lane 2 with lanes 1 and 6). In contrast, stage 2 thymocytes exhibit substantial amounts of partial Dδ1 and Dδ2 to Jδ1 rearrangements and few complete Vδ4 and Vδ5 to DJδ rearrangements (Fig. 1⇑, lane 3); quantitative analysis shows that approximately half of the TCRδ alleles have undergone recombination at this stage. Stage 3 and 4 thymocytes exhibit a TCRδ gene rearrangement pattern similar to that seen in mature T cells (Fig. 1⇑, compare lanes 4 and 5 with lane 9), with the proportion of germline alleles reduced to <5%. The total amount of germline and partial Dδ1 and/or Dδ2-Jδ1 rearrangement hybridization signal is ∼25% (i.e., 75% of the alleles carry complete V-DJδ rearrangements). These results are in sharp contrast to those found for the TCRβ locus, where >95% of the alleles are still in germline configuration at stage 2 10, 12 and only 10–30% of the alleles contain complete V-DJβ rearrangements at stage 3 18 . In summary, it appears that at least half of adult murine intrathymic precursors initiate TCRδ locus recombination at stage 2, when the TCRβ locus is still mainly in germline configuration, and 75–100% have completed V-DJδ rearrangements at stage 3, when only a minority of the cells have full V-DJβ rearrangements. Importantly, because no TCR gene rearrangements are detected in non-T lineage cells 1, 17 , these results argue that irreversible commitment to the T cell lineage occurs as early as stage 2 in many cells.

Analysis of the TCRγ locus reveals an essentially similar pattern of kinetics of V-Jγ rearrangements. Due to a previously unknown polymorphism, this hybridization shows one Vγ7 and two polymorphic Vγ4 germline bands (Fig. 2⇑, lanes 1 and 8). Although the samples shown on lanes 1–6 are all derived from the same group of C57BL/6 mice, the TN subsets were generated from pools of thymi, whereas LN B and T cells were obtained from individual mice. Therefore, the appearance of the two Vγ4 germline bands (tentatively marked as A and B on Fig. 2⇑) varies from lane to lane. However, hybridization to the Vγ4 probe only (data not shown) allows the V to Jγ1 rearrangements to be unambiguously assigned to one of the three upper bands (Fig. 2⇑, compare lane 6 with lane 7). In stage 2 thymocytes, there is a clear appearance of Vγ4-Jγ1 recombination that rapidly increases to a maximum level in stage 3 and 4 cells; this increase is accompanied by a reduction of the corresponding germline hybridization signal. Vγ7 rearrangements are only detectable from stage 3, but their level never reaches the same amount observed for Vγ4 rearrangements (Fig. 2⇑).

PCR analysis of TCRγ and TCRδ gene rearrangements

To analyze TCRγ and TCRδ gene rearrangements with greater sensitivity, genomic DNA from purified TN subpopulations was amplified by PCR using primers designed to detect the three most common adult-type V to DJδ gene rearrangements and the three most common adult-type V to Jγ gene rearrangements. Amplification of a nonrearranging gene (RAG-2) indicates that the input amount of DNA was similar in all of the reactions (see Fig. 3⇓C). The rearrangement of Vγ1 to Jγ4 and Vγ4 to Jγ1 genes (Fig. 3⇓A) and of Vδ4, Vδ5, and Vδ6 genes to Jδ1 (Fig. 3⇓B) can be detected at low levels in stage 2 thymocytes. The signal corresponding to rearranged Vγ and Vδ genes in stage 1 most likely derives from trace numbers of mature γδ contaminants that share the CD24/CD25/CD44 phenotype of stage 1 TN cells 23 . All major Vγ and Vδ genes were found to be rearranged in stages 3 or 4 at levels as high as those seen in total thymocytes (Figs. 3⇓, A and B), indicating that recombination of the majority of TCRγ and TCRδ loci is completed by stage 3. In contrast, analysis of TN subsets shows negligible amounts of TCRβ rearrangements in stage 2 and intermediate levels of V to DJβ recombination at stage 3. Completion of V-DJβ rearrangement to the maximum extent is not seen until more advanced stages. (Fig. 3⇓C, and see Refs. 10, 12, and 18). This analysis further demonstrates that the initiation of V to DJδ and V to Jγ rearrangements precedes V to DJβ recombination. The detection of a few complete TCRγ and TCRδ rearrangements at stage 2 also suggests the possibility that functional γδ TCRs may be generated at this early stage of TN thymocyte differentiation.

A, TCRγ gene rearrangements in TN thymocytes. DNA samples from the four subsets of TN thymocytes (lanes 2–5) and the total thymus (tot, lane 1) were amplified by PCR, Southern blotted, and hybridized with the J gene-specific reverse primers. Rearrangement of the three TCRγ loci most commonly used in adult thymocytes is shown. Note the appearance of Vγ4-Jγ1 and Vγ1-Jγ4 rearrangement in stage 2 TN cells (lane 3). The origin of the background bands in the Vγ1-Jγ4 reactions (upper right panel) is not known, but they are not T cell-specific. B, Rearrangement of the three Vδ genes most commonly used in adult thymocytes in the same samples shown in A. Note the appearance of rearrangement of all three Vδ genes in stage 2 TN cells (lane 3). C, Complete (V8 to DJβ, left panel) and partial (D-Jβ, middle panel) rearrangements of the TCRβ locus in the same samples shown in A. The right panel shows PCR analysis of a nonrearranging gene, RAG-2, visualized by ethidium bromide staining.

Selection of productive TCRγ and TCRδ gene rearrangements

Due to the random, imprecise joining of V, D, and J coding segments, only one-third of TCR gene rearrangements are expected to retain the continuous reading frame with the C domain. Direct sequencing or PCR RFLP analysis 24 can determine the proportion of productive joints within the total pool of rearranged genes. It is expected that any significant deviation from the random 33% distribution of productive joints within a given population of thymocytes results from the effect of a functional TCR on differentiation. Therefore, we performed PCR RFLP analysis for Vδ4, Vδ5, and Vδ6 to Jδ1 rearrangements (Fig. 4⇓A) and Vγ1 to Jγ4 rearrangements (Fig. 4⇓B) on DNA from stage 2–4 TN thymocytes. Vγ7 and Vγ4 rearrangements were not analyzed; Vγ7 rearrangements are not apparent at stage 2 (Fig. 3⇑A), whereas the Vγ4 gene contains an in-frame stop codon at the 3′ end of the gene 25 , which means that productive joints would have to be identified by direct DNA sequencing. The proportion of productive rearrangements of Vδ4/5-Jδ1 and Vγ1-Jγ4 joints in total thymocytes has been shown previously to be significantly less than the random 33% distribution (Fig. 4⇓, and see Refs. 19, 21, and 26). In stage 2 thymocytes, we observed a modest increase from the 33% proportion of in-frame rearrangements for all four joints (Fig. 4⇓, A (lanes 2, 7, and 12) and B (lanes 2 and 7)). Some of these joints are less evenly distributed in stage 2 than in subsequent stages, presumably due to the limited number of independent rearranged alleles present at this early stage. In contrast, there is a reduction in the percentage of productive Vδ4/5-Jδ1 (Fig. 4⇓A, lanes 3, 4, 8, and 9) and Vγ1-Jγ4 (Fig. 4⇓B, lanes 3 and 4) joints in stage 3 and 4 TN thymocytes, although this reduction is less apparent than that observed in the total thymus. The reason for the difference is not known, but may be related to the presence of a recently identified αβ-like thymocyte population among stage 3 and 4 TN cells that is selected for in-frame V-DJδ rearrangements 21, 27 but accounts only for a very small fraction of the total thymus 28 . Despite being selected for productive joints in stage 2 and TCRβ-deficient (mainly γδ lineage) thymocytes, Vδ6-DJδ1 rearrangements are randomly distributed in stage 3 and 4 TN thymocytes as well as in the total thymus 21 . The reason for the lack of underrepresentation of productive joints for these rearrangements in αβ lineage T cells is currently unknown.

A, PCR RFLP analysis of TCRδ gene rearrangements performed on DNA isolated from subsets of TN thymocytes (stages 2–4), a wild-type total thymus (wt total), and a TCRβ-deficient total thymus (β−/− total). The specific Vδ-DJδ1 PCR reactions are shown below each panel. Dashes on the right indicate the position of the productive joints as determined by an analysis of TCRβ−/− thymus samples. B, PCR RFLP analysis of Vγ-Jγ4 rearrangements on the same samples shown in A. C, Summary of the PCR RFLP analyses shown in A and B. The proportion of in-frame Vδ-Jδ and Vγ-Jγ joints among the total rearrangements is shown on the vertical axis. The position of the 33% random distribution is marked with a solid horizontal line.

Because productive TCRγ and TCRδ joints were both enriched at stage 2, it is possible that functional γδ TCRs may positively influence γδ lineage differentiation at this early stage. In support of this concept, surface immunofluorescence staining of CD4−/CD8− thymocytes showed that, although the vast majority of stage 2 cells did not express the γδ TCR, a small fraction of cells (∼1%) are γδ TCR-positive (data not shown). The finding that productive TCRγ and TCRδ gene rearrangements are depleted in stages 3 and 4 further suggests that γδ TCR expression in the previous stages may divert cells into the γδ lineage 26 .

Discussion

Detailed characterization of gene recombination at the various TCR loci in adult thymocytes serves two distinct purposes. First, because TCR gene recombination is a hallmark for T lineage cells 1, 17 , defining the precise point at which TCR gene recombination is initiated would serve as a molecular marker for irreversible T lineage commitment. Second, determining the order of specific TCR rearrangements would be expected to help resolve whether the sequence of these rearrangements might play a role in divergence of the γδ vs αβ lineages, as is implicit in a variety of models 5, 21, 26, 29, 30 . The data contained in this manuscript are relevant to both of these purposes, as is discussed in further detail below.

It has been demonstrated previously that TCRβ rearrangements initiate only during TN stage 3 10, 18 , suggesting that conclusive T lineage commitment does not occur until this point. Functional studies of lineage potential support the exclusive T lineage commitment of stage 3 cells (Ref. 1 and references contained therein). These same studies, however, yielded equivocal results regarding the lineage potential of the earlier stage 2 TN cells 1, 31 . The quantitative Southern blot analysis presented here indicates that more than half of the TCRδ alleles in stage 2 cells show Dδ2 to Jδ1 rearrangements; therefore, by this criteria, at least half of these cells are T lineage-committed. It remains possible that some stage 2 cells have both TCRδ alleles (and, by default, all other TCR loci) in germline configuration. Therefore, we cannot rule out the possibility that cells with multilineage potential (i.e., to T cell and DC or NK cell lineages) persist at this stage. Nonetheless, our findings suggest that the majority of thymocytes commit exclusively to the T lineage by the time they have reached stage 2.

Analysis of the relationship between TCR gene rearrangements and the segregation of αβ vs γδ lineage T cells has fostered two general models of TCR-dependent T lineage divergence 19, 26, 28, 30, 32, 33 , generally referred to as the sequential or competitive models. In the former, a hierarchical rearrangement of TCRγδ genes before that of αβ would lead to the early divergence of γδ T cells, whereas the latter allows that TCRγ, TCRδ, and TCRβ rearrangements may occur simultaneously, with lineage outcome being influenced by whichever receptor (γδ or β/pTα) is expressed first. To help distinguish between these possibilities, we analyzed the order of specific TCRγ and TCRδ gene rearrangements during the defined stages of adult T lymphopoiesis, where TCRβ and TCRα rearrangements have been characterized previously 10, 12, 34, 35 . We find that full recombination of the TCRγ and TCRδ loci can precede that of the TCRβ locus by as much as one developmental stage during adult murine T cell development; recombination of the TCRγ and TCRδ loci initiates during TN stage 2 and is completed by stage 3, whereas V-DJβ rearrangement initiates during TN stage 3 and is completed by stage 4. These results are consistent with the predictions of lineage models in which sequential TCR gene rearrangements influence T lineage divergence. However, it is important to note that recombination of specific TCR loci does not obligate lineage commitment per se, because rearrangement of the TCRγ and TCRδ loci occurs in most αβ lineage T cells 19, 25, 30, 36 , and productive TCRβ rearrangements can be found in γδ T cells 29 . Transgenic experiments have also shown that the TCR cannot exclusively determine commitment to either the γδ or αβ lineages 21, 27, 37, 38 , although it can dramatically influence this commitment 5, 6, 28, 39, 40 . Thus, the experiments described here do not necessarily rule out the possibility that factors other than the TCR can direct lineage commitment, so much as they do support the notion that sequential rearrangements may play a role in this decision. Additional experiments will be required to determine the exact role of TCR-mediated signals in directing commitment among the two T cell lineages.

The finding of an ordered rearrangement of adult TCRγδ vs TCRβ genes reported here parallels the sequential order observed previously in the fetal thymus 33 . However, early fetal TCRγ and TCRδ gene rearrangements are highly restricted with regard to receptor diversity and V gene utilization 41 and show nonrandom junctional sequences irrespective of cellular selection 5 . Fetal thymocytes also differ from their adult counterparts in other aspects of differentiation 8, 42, 43 . Therefore, it is remarkable that although adult thymic precursors use Vγ and Vδ genes that are different from those in early fetal thymocytes (Ref. 2 and references contained therein), the primary accessibility of the TCRδ and TCRγ loci to the VDJ recombinase machinery is retained throughout ontogenesis. Whether distinct waves of Vγ or Vδ gene rearrangements occur in adult thymic precursors, similar to early fetal differentiation 2 , remains to be seen. The data contained here do not reveal striking differences in the usage of various adult-type Vγ and Vδ genes throughout the progression of stages 2 and 3. Although Vγ-Jγ4 and Vγ4-Jγ1 rearrangements appear earlier than Vγ7-Jγ1 rearrangements (see Figs. 2⇑ and 3⇑A), the lesser abundance of Vγ7-Jγ1 rearrangements throughout thymocyte differentiation precludes us from concluding that distinct waves of γδ T cells emerge during adult thymic development. The identification of phenotypically distinct subpopulations of stage 2 and 3 thymocytes will be required to further separate precursors with nonoverlapping rearrangements of the TCRγ, TCRδ, and TCRβ genes.

Acknowledgments

We thank Dr. E. E. Eynon for critical reading of the manuscript. The oligonucleotides used in this study were synthesized by the W. M. Keck Foundation Biotechnology Resource Laboratory at Yale University.

Footnotes

↵1 This work was supported in part by the Howard Hughes Medical Institute and a Presidential Faculty Fellows Award from the National Science Foundation (to D.G.S.) and by National Institutes of Health Grants AI32524 (to D.G.S.), AI33940 and AI/CA39599 (to H.T.P.), and CA08748 (to the Memorial Sloan Kettering Cancer Center).